The present invention relates to methods for determining an effect or effects of a Clostridial toxin or toxins upon a muscle or group of muscles. In particular, the present invention relates to use of dermal topography methods for determining an effect or effects of a Clostridial toxin or toxins upon a facial muscle and for comparing multiple or different Clostridial toxins.
Movement of the face can be due to contractions of muscles underlying the skin and different muscles can move different parts of the face. For example, elevation of the brow results from contraction of the frontalis muscle. Electromyographic methods have been used to study the activity of various facial muscles. See e.g. Fridlund A. et al., Guidelines for Human Electromyographic Research, Psychophysiology 1986; 23(5): 567–590; Vitti M, et al., Electromyographic Investigation of Procerus and Frontalis Muscles, Electromyogr. clin. Neurophysiol. 1976, 16: 227–236, and; Tassinary L. et al., A Psychometric Study of Surface Electrode Placements for Facial Electromyographic Recording: I. The Brow and Cheek Muscle Regions, Psychophysiology 1989; 26(1): 1–16.
In particular, electromyography, including surface electromyography (sEMG) has been used to investigate activity of the frontalis muscle and resultant brow displacement. See e.g. van Boxtel A, et al., Amplitude and bandwidth of the frontalis surface EMG: Effects of electrode parameters, Psychophysiology 1984; 21(6): 699–707, and; Pennock J. D., et al., Relationship between muscle activity of the frontalis and the associated brow displacement, Plast Reconstr Surg November 1999; 104(6): 1789–1797.
Additionally, it is known to study skin topography by making a silicone rubber negative replica (a mold) of a skin surface area. The mold captures three dimensional details of the skin surface and computerized image analysis of skin line density, depths and length analysis shown can be carried out thereon. Grove, G. L., et al, Objective method for assessing skin surface topography noninvasively, chapter one, pages 1–32 of Cutaneous Investigation in Health and Disease, edited by Leveque J-L., Marcel Dekker, Inc. (1989). This method has been used to study how micro-furrows on the forearm can increase in depth from about 33 Φm in children to up to about 100 Φm in the elderly. Corcuff P. et al., Skin relief and aging, J Soc Cosmet Chem 1983; 34:177–190. The same silicone rubber impression method has been used to examine the effect of a topical cream to treat photodamaged skin, as by reduction of periorbital (crow's feet) wrinkles. Leyden J. J., et al., Treatment of photodamaged facial skin with topical tretinoin, J Am Acad Dermatol 1989; 21(3) (part 2): 638–644, and; Grove G. L., et al., Skin replica analysis of photodamaged skin after therapy with tretinoin emollient cream, J Am Acad Dermatol 1991; 25(2) (part 1): 231–237.
Botulinum Toxin
The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals known as botulism. The spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
Botulinum toxin type A is the most lethal natural biological agent known to man. About 50 picograms of botulinum toxin (purified neurotoxin complex) type A (Available from Allergan, Inc., of Irvine, Calif. under the tradename BOTOX®) is a LD50 in mice. One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18–20 grams each. Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F and G each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. The botulinum toxins apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron and blocks the release of acetylcholine.
Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Botulinum toxin type A has been approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus, hemifacial spasm and cervical dystonia. Botulinum toxin type B has also been approved by the FDA for the treatment of cervical dystonia. Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin, type A averages about three months.
Although all the botulinum toxins serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein. Botulinum toxin types B, D, F and G act on vesicle-associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site. Finally, botulinum toxin type C1 has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes.
The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the botulinum toxin type A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C, is apparently produced as only a 500 kD complex. Botulinum toxin type D is produced as both 300 kD and 500 kD complexes. Finally, botulinum toxin types E and F are produced as only approximately 300 kD complexes. The complexes (i.e. molecular weight greater than about 150 kD) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and glutamate.
Botulinum toxin type A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C1, D and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, the botulinum toxin type B serotype only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture.
It has been reported that botulinum toxin type A has been used in clinical settings as follows (“U” and “units” mean the same thing and a used interchangeably):                (1) about 75–250 units of BOTOX® per intramuscular injection (multiple muscles) to treat cervical dystonia;        (2) 5–10 units of BOTOX® per intramuscular injection to treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);        (3) about 30–80 units of BOTOX® to treat constipation by intrasphincter injection of the puborectalis muscle;        (4) about 1–5 units per muscle of intramuscularly injected BOTOX® to treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid.        (5) to treat strabismus, extraocular muscles have been injected intramuscularly with between about 1–5 units of BOTOX®, the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired).        (6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX® into five different upper limb flexor muscles, as follows:                    (a) flexor digitorum profundus: 7.5 U to 30 U            (b) flexor digitorum sublimus: 7.5 U to 30 U            (c) flexor carpi ulnaris: 10 U to 40 U            (d) flexor carpi radialis: 15 U to 60 U            (e) biceps brachii: 50 U to 200 U. Each of the five indicated muscles has been injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX® by intramuscular injection at each treatment session.                        
It is also known that injection of a botulinum toxin into facial muscles can, by weakening the injected muscles, result in a decrease of hyperkinetic wrinkles in the skin overlying the paralyzed muscles. See e.g. Carruthers A. et al., The treatment of glabellar furrows with botulinum A exotoxin, J Dermatol Surg Oncol January 1990;16(1):83.
It is known to use a botulinum toxin to treat: intrathecal pain (see e.g. U.S. Pat. No. 6,113,915); paragangliomas (see e.g. U.S. Pat. No. 6,139,845); otic disorders (see e.g. U.S. Pat. No. 6,265,379); pancreatic disorders (see e.g. U.S. Pat. Nos. 6,143,306 and 6,261,572); migraine (see e.g. U.S. Patent No. 5,714,468); smooth muscle disorders (see e.g. U.S. Patent No. 5,437,291); prostate disorders, including prostatic hyperplasia (see e.g. WO 99/03483 and Doggweiler R., et al Botulinum toxin type A causes diffuse and highly selective atrophy of rat prostate, Neurourol Urodyn 1998; 17(4):363); autonomic nerve disorders, including hyperplasic sweat glands (see e.g. U.S. Pat. No. 5,766,606); wound healing (see e.g. WO 00/24419); reduced hair loss (see e.g. WO 00/62746); skin lesions (see e.g. U.S. Pat. No. 5,670,484), and; neurogenic inflammatory disorders (see e.g. U.S. Pat. No. 6,063,768).
Additionally it has been disclosed that targeted botulinum toxins (i.e. with a non-native binding moiety) can be used to treat various conditions (see e.g. U.S. Pat. No. 5,989,545, as well as WO 96/33273; WO 99/17806; WO 98/07864; WO 00/57897; WO 01/21213; WO 00/10598.
A botulinum toxin has been injected into the pectoral muscle to control pectoral spasm. See e.g. Senior M., Botox and the management of pectoral spasm after subpectoral implant insertion, Plastic and Recon Surg, July 2000, 224–225.
Both liquid stable formulations and pure botulinum toxin formulations have been disclosed (see e.g. WO 00/15245 and WO 74703) as well as topical application of a botulinum toxin (see e.g. DE 198 52 981).
Typically, a Clostridial toxin, such as a botulinum toxin, is administered locally and directly into a target tissue, such as a skeletal muscle, by intramuscular or subcutaneous injection. Entry of a Clostridial toxin into the circulatory system is undesirable, since botulism or tetanus can result. Additionally, entry of a Clostridial toxin into the systemic circulation typically results in generation of antibodies against the toxin. The presence of antibodies leads to a loss or diminishment of a desired clinical response, such as a muscle paralysis. Thus, methodologies for determination of bioavailability of a Clostridial toxin practiced in regard to an intravenously or orally administered pharmaceutical are neither relevant nor applicability with regard to a locally (i.e. intravenous or subcutaneous) administered Clostridial toxin.
Unfortunately, therefore methodologies which examine a physiological fluid (i.e. blood, urine) are of little or no value to determine bioavailability of a Clostridial toxin to a target muscle or muscle group, due to the local (non-systemic) administration and effect of the toxin. Thus, currently available analytical techniques used to perform classical absorption, distribution, biotransformation and elimination studies on an oral or intravenously administered drugs cannot be used.
Botulinum toxin has been injected into facial muscles, such as the orbicularis oculis, corrugator supercilii and frontalis muscles for the cosmetic purpose of reducing certain facial wrinkles, and it is known to use electromyographic and/or photographic techniques to assess the efficacy of such injections. Guerrissi J. et al., Local injection into mimetic muscles of botulinum toxin A for the treatment of facial lines, Ann Plast Surg 1997;39(5):447–53. Electromyography has also been used to assess the effect of injection of a botulinum toxin into the sternocleidomastoid muscle for treatment of cervical dystonia. Dressler D. et al., Electromyographic quantification of the paralysing effect of botulinum toxin in the sternocleidomastoid muscle, Eur Neurol 2000; 43: 13–16. In sEMG the surface electrodes are placed at fixed distances from the injection point, typically 1 cm and 3 cm from the injection point. The surface electrodes can be used to measure the amplitude and area of a compound muscle action potential (CMAP) during maximal voluntary contraction of the injected muscle. One expects to find that CMAP decreases with the onset of muscle paralytic effect and increases as the paralytic effect wears off.
Unfortunately, electromyographic methods for determining an effect of a Clostridial toxin, such as a botulinum toxin, upon a muscle or muscle group can be unsatisfactory because of the variability of electrical activity from a particular muscle between patients an even with the same patient in different positions or on different days due to the known vagaries of electrophysiology. For example, repeat surface electromyographic recordings can show significant (i.e. from about 7% to about 20%) variability when taken from the same patient at the same time. Additionally, the extent of maximal voluntary contraction, at which the sEMG recording is taken, can be variable between and among patients.
Photographic methods, such as digital image analysis, have been used to determine efficacy of a botulinum toxin to treat hyperkinetic facial lines. Heckmann M., et al., Quantification of the efficacy of botulinum toxin type A by digital image analysis, J Am Acad Dermatol 2001; 45: 508–514. As with electromyographic methods, photographic methods also show significant intra and inter subject variability. Thus, photographic methods for determining an effect of a Clostridial toxin, such as botulinum toxin, upon a muscle or a muscle group can lack precision and accuracy and the quality and value of the images obtained are as variant as the lighting conditions, type of film used, film speed and the film development process used.
Thus both electromyographic and photographic methods for assessing an effect of a botulinum toxin upon a muscle have significant drawbacks and deficiencies, and neither of these methods can readily provide a three dimensional permanent record amenable to analysis.
Botulinum Toxin Lethality Assay
The botulinum toxin lethality assay involves the injection of a botulinum toxin composition intraperitoneally into a mouse. For example, the assay may include injecting intraperitoneally (IP) a desired amount of BOTOX® or DYSPORT® into a mouse. Several days after the injection, the injected mice are assessed for mortality. The potency of the product is related to the amount of botulinum toxin that is required to kill 50% of the injected mice (LD50).
Contrary to general belief, the mouse unit (i.e., the amount of botulinum toxin that equals the LD50) is not a standardized unit. It is well documented that the assay to determine the potency of botulinum toxin type A in mouse LD50 units is prone to significant inter-laboratory variability (Schantz and Kautter, J Ass of Anal Chem 1978, 61:96–99). One study designed to standardize a Botulinum type A toxin assay involved 11 different laboratories (Sesardic et al, Pharacol Toxico 1996, 78:283–288). In this study there was found to be up to a 10-fold difference in results. This variability in mouse LD50 is not unique to assays involving botulinum toxin. In fact, because of the variability of this assay, a number of regulatory agencies have abandoned requiring the routine use of LD50 for toxicity testing for a number of chemicals, solvents, cosmetics and drugs (Pearce et al, Toxicol App Pharm 1994, 128:69–77; U.S. Pat. Nos. 5,401,243 and 5,183,462,).
In addition, although BOTOX® and DYSPORT® are both botulinum toxin type A-containing compositions, BOTOX® has a greater potency than DYSPORT® when therapeutically assessed. However, when measuring the potency of BOTOX® and DYSPORT® using the lethality assay described above, BOTOX® and DYSPORT® exhibit similar potencies.
The expanding medical importance of botulinum toxins has increased the need for, and placed a premium on, the precise analysis of biological activity contained in preparations of botulinum toxin type A for both clinical use and laboratory investigation.
What is needed therefore is a non-invasive method for determining a pharmacodynamic effect (such as a muscle paralytic effect) of a Clostridial toxin, such as a botulinum toxin, upon a muscle or muscle group, which method provides an accurate and precise three dimensional record amendable to computerized analysis. There also remains a need for a non-invasive and non-lethal methods for determining the potency of a Clostridial toxin, and for methods of comparing neurotoxins.